Lignans are secondary metabolites (e.g., sesamin, sesamolin, etc.) in vascular plants and widely distributed in plants. It is reported so far that lignans are contained in the seeds, fruits, cut panicles, tubers and/or tuberous roots, etc. of plants. Lignans are considered to contribute mainly to biological defense mechanisms in plants. In addition to plants, lignans have attracted attention since they have a wide variety of physiological and pharmacological functions in organisms other than plants because of their potent antioxidative effects or the like. Lignans have the structure in which two phenylpropanoid molecules having the C6-C3 skeleton are dimerized, and those with an 8,8′ linkage are the most prevalent class of lignans (cf., Lignans, D. C. Ayres and J. D. Loike (1990)).
Representative lignans include (+)-pinoresinol, (+)-sesamin, (+)-sesaminol, (+)-sesamolin and (+)-sesamolinol contained in sesame (Sesamum indicum); (+)-pinoresinol, (−)-arctigenin and (−)-matairesinol contained in forsythia (Forsythia intermedia); (−)-pinoresinol and (−)-lariciresinol contained in Daphne tangutica; (+)-secoisolariciresinol contained in linum (Linum usitatissimum); etc. Molecular structures of these lignans are diverse and classified into 8 subclasses in view of the skeletal structure (cf., Phytochemistry Rev. (2003) 2: 371-390).
Biosynthetic pathways of lignans have been studied, focusing mainly on Sesamum indicum of the family Pedaliaceae, Forsythia intermedia of the family Oleaceae and Linum usitatissimum of the family Linaceae. Some enzymes and genes which catalyze the metabolic pathways are reported. It is reported that among them, pinoresinol synthesized by polymerization of coniferyl alcohol is the first lignan in the biosynthesis and a variety of lignans are synthesized from pinoresinol via biosynthetic pathways specific to individual plant species (cf., J. Wood., Sci. 53, 273-284 (2007), Lignans: biosynthesis and function, Comprehensive natural products chemistry, (1999) 1: 640-713). Piperitol is synthesized by the action of piperitol synthase on (+)-pinoresinol. Pinoresinol is the lignan synthesized at the earliest stage of lignan biosynthetic pathways and therefore is the major lignan distributed in many plants, for example, in plants of the families Asteraceae, Oleaceae, Compositae, Umbelliferae, Thymelaeaceae, Magnoliaceae, Liliaceae and Pinaceae.
As enzymes involved in the biosynthesis of lignans, dirigent proteins which take part in pinoresinol synthesis are reported on Forsythia intermedia, etc. (cf., Non-Patent Literature 6: Plant Physiol., (2000) 123: 453 and Patent Literature 1: Japanese National Publication (Tokuhyo) No. 2001-507931, etc.). As genes for enzymes involved in the lignan biosynthesis and their utilization, there are further reports on the gene for pinoresinol-lariciresinol reductase in Forsythia intermedia (cf., Non-Patent Literature 7: J. Biol. Chem., (1996) 271: 29473 and Patent Literature 1), on the gene for pinoresinol-lariciresinol reductase in Thuja plicata (cf., Non-Patent Literature 8) and recombinant secoisolariciresinol dehydrogenase and the method of its use (cf., Non-Patent Literature 9: J. Biol. Chem., (2001) 276: 12614, Patent Literature 2: Japanese National Publication (Tokuhyo) No. 2002-512790, etc.). Furthermore, the gene for cytochrome P450 enzyme having a piperitol-sesamin synthetic activity from Sesamum indicum and its use are reported (cf., Non-Patent Literature 10: Proc. Nat. Acad. Sci. USA, (2006) 103: 10116 and Patent Literature 3: Japanese National Publication (Tokuhyo) No. 2007-507201).
Lignans are known to undergo a variety of modifications including glycosidation, hydroxylation, methylation, prenylation, etc., after the skeletal formation. The genes for glycosyltransferases having a glycosidation activity on furofuran lignans such as sesaminol, which is one of the sesame lignans, are isolated and their use is reported (cf., Patent Literature 1: Japanese Laid-Open Patent Publication (Tokkai) No. 2006-129728).
It is known that 9-hydroxy derivatives of furofuran lignans represented by pinoresinol are present in Ligularia kanaitizensis belonging to the genus Ligularia of the Asteraceae family or Allamanda neriifolia belonging to the genus Allamanda of the family Compositae, which are native to China (cf., Non-Patent Literature 1: Lignans, D. C. Ayres and J. D. Loike (1990); Non-Patent Literature 2: Phytochemistry, (1988) 27, 575; Non-Patent Literature 3: Indian J. Chem. (1995) 34B, 975, etc.). It is reported that 9-hydroxylpinoresinol which is one of 9-hydroxy derivatives has an antioxidative and butyrylcholinesterase activity in an in vivo test, and recently a neuroprotective effect against oxidative damages in the brain (Non-Patent Literature 4: J. Pharmacy and Pharmacology, (2005) 57, 233; Non-Patent Literature 5: Proceedings of the Pakistan Academy of Sciences, (2005) 42, 167; Non-Patent Literature 6: J. Pharm. Pharmacol., (2007) 59: 521, etc.). It is also reported that an anti-HIV-1 reverse transferase (RT) activity is noted for 2-methyl-2-butenoicpinoresinol obtained by further modification of the hydroxy to the ester, and clarification of the biosynthetic pathway is expected (cf., Non-Patent Literature 4: J. Pharmacy and Pharmacology, (2005) 57, 233; Non-Patent Literature 5: Proceedings of the Pakistan Academy of Sciences, (2005) 42, 167; Non-Patent Literature 6: J. Pharm. Pharmacol., (2007) 59: 521, etc.).
Notwithstanding that the utility of lignans having hydroxy is reported as stated above, (+)-larreatricin hydroxylase isolated from chaparral of the family Zygophyllaceae is the only lignan hydroxylase (cf, Non-Patent Literature 7: Proc. Nat. Acad. Sci. USA, (2006) 100: 10641). This enzyme belongs to the polyphenol oxidase (PPO) family, which is one of oxidases in plants (cf., Non-Patent Literature 8: Trends in Plant Science, (2007) 12, 29). In addition to this enzyme family, the oxidase families such as cytochrome P450 enzyme, 2-oxoglutarate-dependent oxygenase, etc. are also known to catalyze the hydroxylation of lignans as well (cf., Non-Patent Literature 9: De Montellano, P. R. O., Cytochrome P450-structure, mechanism, and biochemistry. 3rd edition. Kluwer Academic/Plenum Publishers, NY, (2005) and Non-Patent Literature 10: J. Biol. Chem. (2004) 279, 1206). However, (+)-larreatricin hydroxylase is a hydroxylase for lignans having no oxygen at position 9 (9′) of the furan ring, but any enzyme that may catalyze the hydroxylation at position 9 of furofuran lignans represented by pinoresinol still remains unclear. Thus, it has been further desired to acquire genes for lignan oxidase and analyze their functions.    Patent Literature 1: Japanese Laid-Open Patent Publication (Tokkai) No. 2006-129728    Non-Patent Literature 1: Lignans, D. C. Ayres and J. D. Loike (1990)    Non-Patent Literature 2: Phytochemistry, (1988) 27, 575    Non-Patent Literature 3: Indian J. Chem., (1995) 34B, 975    Non-Patent Literature 4: J. Pharmacy and Pharmacology, (2005) 57, 233    Non-Patent Literature 5: Proceedings of the Pakistan Academy of Sciences, (2005) 42, 167    Non-Patent Literature 6: J. Pharm. Pharmacol., (2007) 59: 521    Non-Patent Literature 7: Proc. Nat. Acad. Sci. USA, (2003) 100: 10641    Non-Patent Literature 8: Trends in Plant Science, (2007) 12, 29    Non-Patent Literature 9: De Montellano, P. R. O., Cytochrome P450-structure, mechanism, and biochemistry, 3rd edition, Kluwer Academic/Plenum Publishers, NY. (2005)    Non-Patent Literature 10: J. Biol. Chem. (2004) 279, 1206